The machinery responsible for making proteins (e.g. ribosomal RNA, ribosomal proteins, translation factors, and tRNAs) is central to growth and development of all organisms, and the control of its synthesis has been a central issue in molecular microbioogy for over 50 years. More recently, it has also become clear that an understanding of the mechanisms responsible for rRNA and tRNA transcription in Escherichia coli can provide fundamental insights into the mechanism of transcription in general. The questions addressed in the proposal are divided into five specific aims. In the first aim, we will continue to study the role of the C-terminal domain of the alpha subunit of RNA polymerase in bacterial promoter function, specifically how it stimulates isomerization steps in the process of transcription initiation, as well as its role in activation by the transcription factor Fis. In the second aim, we propose to determine how the so-called Discriminator Region (the part of the promoter just downstream of the -10 element) interacts with RNAP to regulate transcription and to determine the identity of the transcription start site. In the third aim, we propose to determine the sites of interaction of RNAP with ppGpp and DksA, two molecules crucial for regulation of bacterial transcription initiation, in order to gain insights into mechanisms of transcription regulation by small molecules. In the fourth aim, we propose to determine other promoters regulated by ppGpp, DksA, and the concentration of the initial NTP in the transcript and thus to uncover previously-unsuspected connections between the synthesis of the translation machinery and the synthesis of the machineries for other cellular processes. In the final aim, we propose to determine the structure and mechanism of Crl, a small protein that we have recently proposed might function as a sigma subunit chaperone. Since many of these factors are also virulence factors, regulating expression of genes that play important roles in bacterial pathogenesis, it has become clear in recent years that these studies are important not only for understanding the mechanism of transcription initiation in general, a fundamental step in gene expression in all organisms, but also for an understanding of infectious disease and its prevention.